Prothymosin alpha is a highly acidic, nuclear protein which is known to accumulate in proliferating cells. Its function has been investigated by means of four different antisense oligodeoxyribonucleotides directed toward different regions of prothymosin alpha mRNA. In every case, when synchronized human myeloma cells were released from stationary phase by incubation in fresh medium containing antisense oligomers, cell division was prevented or inhibited. The sense counterparts of these oligomers had no effect. A detailed analysis of antisense-treated cells indicated that growth inhibition was reversible; the cells divided one cell division late relative to sense-treated or untreated myeloma cells. The failure to divide correlated directly with a deficit in prothymosin alpha and with the survival, primarily in the nucleus, of intact antisense oligomers. Our data suggest that prothymosin alpha is essential for cell division. The two forms of prothymosin alpha mRNA were characterized and found to arise by alternative splicing of one functional gene. As a result of nonconsensus splice acceptor selection, a GAG codon, which would be expected to be exonic, is sequestered within the adjacent intron to generate the common form of prothymosin a mRNA. The rare form (0.5% of prothymosin a mature mRNA in placenta) contains the GAG codon and encodes an identical protein made one residue longer by the insertion of glutamic acid at position 40 of the protein. This is the first example of alternative splicing in which one gene, without adding, subtracting, or substituting exons, gave rise to two almost identical transcripts, with the larger acquiring an triplet introduced, in frame, into sequences specifying the fully processed protein. Reverse transcriptase from human immunodeficiency virus uses one DNA:RNA binding site for polymerization of complementary DNA on RNA templates or for hydrolysis of RNA in DNA:RNA hybrids.